DNA-induced liquid phase condensation of cGAS activates innate immune signaling

The binding of DNA to cyclic GMP-AMP synthase (cGAS) leads to the production of the secondary messenger cyclic GMP-AMP (cGAMP), which activates innate immune responses. Here, we show that DNA binding to cGAS robustly induced the formation of liquid-like droplets in which cGAS was activated. The disordered and positively charged cGAS N-terminus enhanced cGAS–DNA phase separation by increasing the valencies of DNA binding. Long DNA was more efficient in promoting cGAS liquid phase separation and cGAS enzyme activity than short DNA. Moreover, free zinc ion enhanced cGAS enzyme activity both in vitro and in cells by promoting cGAS–DNA phase separation. These results demonstrated that the DNA-induced phase transition of cGAS promotes cGAMP production and innate immune signaling.

To establish the BJ-5ta-Halo-cGAS cell line, BJ-5ta cells were infected with lentiviruses carrying Halo-hcGAS-Flag and a neomycin-resistance gene. After neomycin (400 µg/ml) selection, surviving cells were frozen or passaged for studies.
To establish MEF cGAS KO -GFP-cGAS and MEF cGAS KO -GFP-DN160cGAS cell lines, primary MEF cGAS KO cells were isolated from C57BL/6 cGas -/mice and immortalized by SV40 T antigen transformation. Immortalized MEF cGAS KO cells were infected with lentiviruses carrying GFP-cGAS or GFP-DN160cGAS and a puromycin-resistance gene. After puromycin (1 µg/ml) selection, surviving cells were frozen or passaged for studies.
The His 6 -SUMO tag was cleaved by SUMO protease (Ulp1) at 4 ℃ overnight. Cleaved protein was applied to a 1-ml HiTrap Heparin column (GE Healthcare). After washing with 20 mM Tris-HCl, pH 7.5, 500 mM NaCl, cGAS protein was eluted with a gradient of 0.5-1 M NaCl in 20 mM Tris-HCl, pH 7.5. Eluted cGAS protein was subjected to size exclusion chromatography using a Superdex 200 column (GE Healthcare) in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl and the fractions were collected, concentrated, and dialysed against a buffer containing 20 mM Tris-HCl, pH 7.5 and 150 mM NaCl. Recombinant cGAS protein was labeled with Alexa Fluor 488 by using Alexa Fluor™ 488 Protein Labeling Kit (ThermoFisher) and the estimated degree of labeling was 2 moles of Alexa Fluor 488 per mole of recombinant cGAS.

In vitro Phase Separation Assay
In general, recombinant cGAS protein (3% Alexa Fluor 488-labeled) was mixed with DNA of a defined length (2% Cy3-labeled) in 96-well plates (Corning) coated with 20 mg/ml BSA (Sigma). Mixtures were incubated and images were captured at indicated times. Phase separation of recombinant cGAS with 45-bp ISD was performed in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl and 1 mg/ml BSA. Phase separation of recombinant cGAS with 100-bp DNA was performed in 20 mM Tris-HCl, pH 7.5, 300 mM NaCl, 1 mg/ml BSA. Phase diagrams were generated by mixing recombinant cGAS with 45-bp DNA or 100-bp DNA in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl and 1 mg/ml BSA. Phase separation with 45-bp dsRNA was performed in physiological buffer (20 mM Tris-HCl, pH 7.5, 15 mM NaCl, 135 mM KCl, 5 mM Phosphate, 1.5 mM MgCl 2 , and 1 mg/ml BSA). Phase separation in the presence or absence of zinc (200 µM ZnCl 2 ) was performed by mixing Alexa Fluor 488-labeled cGAS with Cy3-labeled DNA in physiological buffer.

Image Acquisition and Analysis
Phase separated droplets were imaged by using Nikon A1R+ confocal microscope with 40× oil objective, Nikon A1 camera, and X-Cite 120LED laser. Imaging power was 0.5% and images were analyzed using ImageJ (Open source Java program from NIH). Time lapse images of cGAS-DNA phase separation were captured using the Time Lapse Acquisition tool of a Nikon A1R+ confocal microscope every 20 seconds over 2 hours. Fluorescence intensities and EqDiameter (the diameter of a circle with the same area as the measured object) of phase separated droplets were quantified using Nikon NIS-Elements AR (Advanced Research) software. The distribution of droplets EqDiameter was plotted using GraphPad Prism 7.

In vitro FRAP Assays
Fluorescence recovery after photobleaching (FRAP) experiments were performed on a Nikon A1R+ confocal microscope at either 25 ℃ or 37 ℃. For FRAP of cGAS or DNA, spots of ~2-μm diameter in ~ 10-μm droplets were photobleached with 20% laser power for 1 second using 488nm and 561-nm lasers. Time lapse images were acquired over a 20-min time course after bleaching with 10 s interval. For FRAP of ATP, droplets with diameter in ~ 5-μm was fully photobleached with 100% laser power for 10 seconds using 640-nm laser. Time lapse images were acquired over a 1-min time course after bleaching with 1.2 s interval. Images were processed by ImageJ. Fluorescence intensities of regions of interest (ROIs) were corrected by unbleached control regions and then normalized to pre-bleached intensities of the ROIs. The corrected and normalized data were fit to the single exponential model by GraphPad Prism 7: where % was the intensity at the start of recovery after bleaching, ( was the plateau intensity, and was the exponential constant. was calculated by the reciprocal of . The recovery rate was calculated by ( divided by the fluorescence intensity before bleaching. 3/5 represents the time point achieving half recovery intensity ( 6 7 -6 8 5 ).

Cellular FRAP Assays
Cellular fluorescence recovery after photobleaching (FRAP) experiments were performed on a Nikon A1R+ confocal microscope at 37 ℃ in a live-cell-imaging chamber. MEF cGAS KO -GFP-cGAS cells were grown on chambered cover glass until it reached the desired density, at which time cells were transfected with 45-bp Cy5-labeled ISD for 4 hours using lipofectamine 2000.
cGAS-DNA puncta were fully or partially photobleached with 20% laser power for 2 s using a 488-nm laser. Time-lapse images were acquired over a 5-min time course after bleaching with 10-second interval. Images were processed by ImageJ and FRAP data were fit to a single exponential model by GraphPad Prism 7.

Live Cell Imaging
Imaging of cGAS-DNA puncta and zinc ions in BJ-5ta-Halo-cGAS cells Cells were grown on chambered cover glass to a proper density and then incubated with 5 µM cell-permeant HaloTag TMR ligand (Promega) in the culture medium at 37 ℃ for 15 min. The cells were rinsed three times with PBS and incubated in culture medium at 37 ℃ for 30 min.
After labeling, cells were transfected with fluorescein-labeled DNA using lipofectamine 2000.
Live cell images were captured after 2 hours by using Nikon A1R+ confocal microscope with 40× oil objective, Nikon A1 camera, and X-Cite 120LED laser. Images were analyzed by ImageJ. For detection of intracellular zinc, TMR-labeled BJ-5ta-Halo-cGAS cells were transfected with Cy5-ISD for 2 hours and then rinsed twice with 1× Tyrode's solution (139 mM NaCl, 3 mM KCl, 17 mM NaHCO 3 ,12 mM Glucose, 3 mM CaCl 2 , and 1 mM MgCl 2 ). Cells were incubated with 5 µM Zinpyr-1 (AdipoGen) in 1× Tyrode's solution at room temperature for 10 min, followed by rinsing with the same solution for 10 min. Cell images were captured and analyzed as above.

cGAS-DNA puncta imaging with MEF cGAS KO -GFP-cGAS cells or MEF cGAS KO -GFP-DN160cGAS
cells MEF cGAS KO -GFP-cGAS or MEF cGAS KO -GFP-DN160cGAS cells growing on chambered cover glass were transfected with Cy5-labeled ISD with lipofectamine 2000, and confocal microscopy was performed and analyzed as described above. For time-lapse live-cell imaging, images were acquired over a 4-hour time course with 5-min intervals, followed by data processing using ImageJ.

Saponin Permeabilization Assay
MEF cGAS KO -GFP-cGAS cells growing on chambered cover glass were transfected with 45 bp Cy5-ISD by lipofectamine 2000 for 4 hours. After washing twice with PBS, cells were stained with plasma membrane dye (5 µM Wheat Germ Agglutinin, Alexa Fluor 350 Conjugate, ThermoFisher) for 10 min. Then cells were washed twice with PBS and incubated with 0.03% saponin in PBS at room temperature for 3 min. Cells were washed with PBS and fixed with 4% paraformaldehyde (Electron Microscopy Sciences) in PBS for 10 min. After two more washes in PBS, cells were imaged using Nikon A1R+ confocal microscope with 40× oil objective, Nikon A1 camera, and X-Cite 120LED laser. Images were analyzed by ImageJ.

cGAS Activity Assay and cGAMP Measurement
In vitro cGAS reaction was performed by mixing recombinant human or mouse cGAS protein with ATP (5 mM), GTP (300 µM) and HT-DNA (15 ng/µl) in either the low-salt buffer (20 mM Tris-HCl pH 7.5, 5 mM MgCl 2 , and 0.2 mg/ml BSA) or the physiological buffer (20 mM Tris-HCl, pH 7.5, 15 mM NaCl, 135 mM KCl, 5 mM Phosphate, 1.5 mM MgCl 2 , and 0.2 mg/ml BSA). After incubation at 37 ℃ for 2 hours, reaction was terminated by heating at 95 ℃ for 5 min to denature proteins, which were removed by centrifugation at 20,000 × g for 5 min. The supernatant was delivered into THP1-Lucia ISG cells (0.25 × 10 6 in a 50-µl reaction) that were permeabilized with perfringolysin O (PFO; 50 ng/ml). The cells were cultured at 37 ℃ for 16 h, at which time the secreted luciferase activity in the culture media was measured using CLARIOstar (BMG LABTECH). Different concentrations of cGAMP were used to generate the standard curve for estimating cGAMP concentrations in the reactions.
The cGAMP bioassay described above was also used to measure cGAMP levels in DNAtransfected cells. Cells were transfected with ISD or HT-DNA by lipofectamine 2000 in Opti-MEM medium. Four hours after transfection, cells were harvested and lysed in 50 µl of hypotonic buffer (10 mM Tris-HCl, pH7.5, 5 mM KCl, and 3 mM MgCl 2 ) supplemented with protease inhibitor cocktail. Cell lysates were heated at 95 ℃ for 5 min to denature proteins, which were precipitated by centrifugation. The supernatant containing cGAMP was measured by delivering into THP1-Lucia cells as described above.

Subcellular fractionation
Hela or THP1 cells were transfected with HT-DNA by lipofectamine 2000 in Opti-MEM medium for 2 hours before cells (5 × 10 7 ) were lysed by passing through a 30G1 needle five times in 500 µl hypotonic buffer (10 mM Tris-HCl, pH 7.5, 5 mM KCl, and 3 mM MgCl 2 ) supplemented with a protease inhibitor cocktail. The homogenate was centrifuged at 2,000 × g for 10 min and the pellet (P2) was washed with hypotonic buffer and resuspended into a desired buffer for analysis or further fractionation by ultracentrifugation. The supernatant (S2) was centrifuged at 20,000 × g for 10 min and the pellet (P20) was washed with hypotonic buffer and resuspended into a desired buffer for analysis. The supernatant was collected as S20 for analysis.
P2 fractionation by Optiprep gradient and cGAS activity assay P2 pellet was resuspended in 200 µl of 17.5 % Optiprep with Iso-osmotic Buffer (20 mM Tris-HCl, pH 7.5, 250 mM Sucrose, 25 mM KCl, and 5 mM MgCl 2 ). Optiprep solutions at different densities (20%, 22.5%, 25%, 27.5%, 30%, 32.5%, and 35% in Iso-osmotic buffer) were prepared and used to generate a discontinuous gradient by layering 200 µl of each solution on top of each other from higher density (bottom) to lower density (top) in a centrifuge tube. Then, 200 µl of the P2 fraction was loaded on top of the layered gradient and the tubes were subjected to ultracentrifugation at 100,000 × g for 2 h. After ultracentrifugation, 100 µl of each fraction was removed by pipette carefully from top to bottom. The total fraction number was 16.
Ten microliters of each fraction was mixed with ATP (5 mM) and GTP (500 µM) in the physiological buffer (20 mM Tris-HCl, pH 7.5, 15 mM NaCl, 135 mM KCl, 5 mM Phosphate, 10 mM MgCl 2 , 400 µM ZnCl 2 and 0.2 mg/ml BSA). The reaction mixture with a total volume of 60 µl was incubated at 37 ℃ for 2 hours, followed by heat inactivation at 95 ℃ for 5 min. After centrifugation at 20,000 × g for 5 min, supernatant was delivered into THP1-Lucia ISG cells to measure the cGAMP levels. The cGAMP produced inside Hela or THP1 cells during DNA transfection was theoretically fractionated into S20 (here we refer to as endogenous cGAMP), and the endogenous cGAMP production was measured and subtracted.

Zinc Chelation in Cells
L929 cells growing in six-well tissue culture plates were incubated with the zinc chelator TPEN at different concentrations for 2 hours before cells were transfected with HT-DNA by lipofectamine 2000 in DMEM medium (zinc free). Two hours after transfection, cells were lysed in 200 µl of hypotonic buffer (10 mM Tris-HCl, pH7.5, 5 mM KCl, and 3 mM MgCl 2 ) containing a protease inhibitor cocktail. cGAMP in the cell lysates was measured as described above.

cGAS Thermo Shift Assay
The recombinant hcGAS-FL protein was mixed with or without HT-DNA or ZnCl 2 (200 µM) in the Protein Thermal Shift Buffer (ThermoFisher) supplemented with a Protein Thermal Shift Dye (1000×) (ThermoFisher). Fluorescence reporter signals were recorded on a ViiA7 Real-Time PCR System (Applied Biosystems) with its Melt Curve option and ROX reporter type with Excitation Filter-Emission Filter x4 (580 ± 10)-m4 (623 ± 14) nm. Samples were subjected to a temperature gradient in the PCR machine from 25 ℃ to 99 ℃ with the Ramp rate of 0.05 ℃/s. The T m values were determined by fitting the melting curves to a Boltzmann sigmoidal equation using GraphPad Prism 7 software.

Measurement of zinc ion binding to cGAS or DNA
10 μM ZnCl 2 was incubated with various concentrations of DNA, cGAS, or both in a buffer containing 20 mM Tris-HCl, pH 7.5 and 150 mM NaCl. The mixture was passed through a 0.5 ml centrifugal filter with 30 kDa cutoff (Amicon), which was pre-rinsed with a buffer containing 9 20 mM Tris-HCl pH 7.5, 150 mM NaCl, and 10 μM ZnCl 2 . After centrifuging the filters at 12,000 × g for 2 min, zinc ion concentration in the filtrate was measured by using a zinc quantification kit (Abcam). The data points were fitted to the equation of specific binding with Hill Slope in GraphPad Prism7. Kd is the cGAS concentration needed to achieve a halfmaximum binding at equilibrium.

Estimation of Cytoplasmic cGAS Concentration
The cytoplasmic cGAS concentration of Hela cells was estimated by two methods as follows: A. The proteins concentration of Hela cytoplasm is about 100 mg/ml (28)